Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50 = 20 µM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

Sublimation of isolated toric focal conic domains on micro-patterned surfaces.

Toric focal conic domains (TFCDs) in smectic liquid crystals exhibit distinct topological characteristics, featuring torus-shaped molecular alignment patterns with rotational symmetry around a central core. TFCDs have attracted much interest due to their unique topological structures and properties, enabling not only fundamental studies but also potential applications in liquid crystal (LC)-based devices. Here, we investigated the precise spatial control of the arrangement of TFCDs using micropatterns and sublimation of TFCDs to estimate the energy states of the torus-like structures. Through simulations, we observed that the arrangement of TFCDs strongly depends on the shape of the topographies of underlying substrates. To accurately estimate the energetic effects of non-zero eccentricity and evaluate their thermodynamic stability, we propose a geometric model. Our findings provide valuable insights into the behavior of smectic LCs, offering opportunities for developing novel LC-based devices with precise control over their topological properties.

Variation of C-terminal domain governs RNA polymerase II genomic locations and alternative splicing in eukaryotic transcription.

The C-terminal domain of RPB1 (CTD) orchestrates transcription by recruiting regulators to RNA Pol II upon phosphorylation. Recent insights highlight the pivotal role of CTD in driving condensate formation on gene loci. Yet, the molecular mechanism behind how CTD-mediated recruitment of transcriptional regulators influences condensates formation remains unclear. Our study unveils that phosphorylation reversibly dissolves phase separation induced by the unphosphorylated CTD. Phosphorylated CTD, upon specific association with transcription regulatory proteins, forms distinct condensates from unphosphorylated CTD. Function studies demonstrate CTD variants with diverse condensation properties in vitro exhibit difference in promoter binding and mRNA co-processing in cells. Notably, varying CTD lengths lead to alternative splicing outcomes impacting cellular growth, linking the evolution of CTD variation/length with the complexity of splicing from yeast to human. These findings provide compelling evidence for a model wherein post-translational modification enables the transition of functionally specialized condensates, highlighting a co-evolution link between CTD condensation and splicing.

Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirABVp toxin produced by Vibrio parahaemolyticus.

Introduction: The binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirABVp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirABVp. Methods: We utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirABVp. Results: Antibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (KD), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirABVp. The developed immunoassay detected PirABVp in the protein lysates of AHPND-causing V. parahaemolyticus (VpAHPND) and showed a significant detectability in moribund or dead shrimp infected with a VpAHPND virulent strain compared to that in non-infected shrimp. Discussion: These results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirABVp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified VpAHPND in the global shrimp culture industry.

Efficient Fluoride Wastewater Treatment Using Eco-Friendly Synthesized AlOOH.

Fluoride ion is essential for health in small amounts, but excessive intake can be toxic. Meeting safety regulations for managing fluoride ion emissions from industrial facilities with both cost-effective and eco-friendly approaches is challenging. This study presents a solution through a chemical-free process, producing a boehmite (AlOOH) adsorbent on aluminum sheets. Utilizing cost-effective Al foil and DI water, rather than typical precursors, yields a substantial cost advantage. The optimized AlOOH adsorbent demonstrated a high fluoride ion removal rate of 91.0% in simulated wastewater with fluoride ion concentrations below 20 ppm and displayed a similar performance in industrial wastewater. Furthermore, the AlOOH adsorbent exhibited excellent reusability through a simple regeneration process and maintained stable performance across a wide pH range of 4 to 11, demonstrating its capability to adsorb fluoride ions under diverse conditions. The efficiency of the AlOOH adsorbent was validated by a high fluoride ion removal efficiency of 90.9% in a semi-batch mode flow cell, highlighting its potential applicability in engineered water treatment systems. Overall, the AlOOH adsorbent developed in this study offers a cost-effective, eco-friendly, and sustainable solution for effectively removing fluoride ion from surface waters and industrial wastewaters.

Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations.

The key to type 1 copper (T1Cu) function lies in the fine tuning of the CuII/I reduction potential (E°'T1Cu ) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tune E°'T1Cu in azurin over a wide range, these principles are yet to be generalized to other T1Cu-containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) from Streptomyces coelicolor to match the high E°'T1Cu of fungal laccases. Using ultraviolet-visible absorption and electron paramagnetic resonance spectroscopies, together with X-ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and corresponding E°'T1Cu . While minimal and small E°'T1Cu increases are observed in Y229F- and S292F-SLAC, the V290N mutant exhibits a major E°'T1Cu increase. Moreover, the influence of these mutations on E°'T1Cu is additive, culminating in a triple mutant Y229F/V290N/S292F-SLAC with the highest E°'T1Cu of 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (kcat /KM ) relative to WT-SLAC.

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue.

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering thermogenesis. To achieve this, BAT utilizes a diverse menu of circulating nutrients to support its high metabolic demand. Additionally, BAT secretes metabolite-derived bioactive factors that can serve as either metabolic fuels or signaling molecules, facilitating BAT-mediated intratissue and/or intertissue communication. This suggests that BAT actively participates in systemic metabolite exchange, an interesting feature that is beginning to be explored. Here, we introduce a protocol for in vivo mouse-level optimized BAT arteriovenous metabolomics. The protocol focuses on relevant methods for thermogenic stimulations and an arteriovenous blood sampling technique using Sulzer's vein, which selectively drains interscapular BAT-derived venous blood and systemic arterial blood. Next, a gas chromatography-based metabolomics protocol using those blood samples is demonstrated. The use of this technique should expand the understanding of BAT-regulated metabolite exchange at the inter-organ level by measuring the net uptake and release of metabolites by BAT.

Emerging role of Hippo pathway in the regulation of hematopoiesis.

In various organisms, the Hippo signaling pathway has been identified as a master regulator of organ size determination and tissue homeostasis. The Hippo signaling coordinates embryonic development, tissue regeneration and differentiation, through regulating cell proliferation and survival. The YAP and TAZ (YAP/TAZ) act as core transducers of the Hippo pathway, and they are tightly and exquisitely regulated in response to various intrinsic and extrinsic stimuli. Abnormal regulation or genetic variation of the Hippo pathway causes a wide range of human diseases, including cancer. Recent studies have revealed that Hippo signaling plays a pivotal role in the immune system and cancer immunity. Due to pathophysiological importance, the emerging role of Hippo signaling in blood cell differentiation, known as hematopoiesis, is receiving much attention. A number of elegant studies using a genetically engineered mouse (GEM) model have shed light on the mechanistic and physiological insights into the Hippo pathway in the regulation of hematopoiesis. Here, we briefly review the function of Hippo signaling in the regulation of hematopoiesis and immune cell differentiation. [BMB Reports 2023; 56(8): 417-425].

Structural Basis for the Effects of Phenylalanine on Tuning the Reduction Potential of Type 1 Copper in Azurin.

In order to investigate the effects of the secondary coordination sphere in fine-tuning redox potentials (E°') of type 1 blue copper (T1Cu) in cupredoxins, we have introduced M13F, M44F, and G116F mutations both individually and in combination in the secondary coordination sphere of the T1Cu center of azurin (Az) from Pseudomonas aeruginosa. These variants were found to differentially influence the E°' of T1Cu, with M13F Az decreasing E°', M44F Az increasing E°', and G116F Az showing a negligible effect. In addition, combining the M13F and M44F mutations increases E°' by 26 mV relative to WT-Az, which is very close to the combined effect of E°' by each mutation. Furthermore, combining G116F with either M13F or M44F mutation resulted in negative and positive cooperative effects, respectively. Crystal structures of M13F/M44F-Az, M13F/G116F-Az, and M44F/G116F-Az combined with that of G116F-Az reveal these changes arise from steric effects and fine-tuning of hydrogen bond networks around the copper-binding His117 residue. The insights gained from this study would provide another step toward the development of redox-active proteins with tunable redox properties for many biological and biotechnological applications.

Fabrication of Scratched Nanogrooves for Highly Oriented Cell Alignment and Application as a Wound Healing Dressing.

Using improper wound care materials may cause impaired wound healing, which can involve scar formation and infection. Herein, we propose a facile method to fabricate a cell-alignment scaffold, which can effectively enhance cell growth and migration, leading to the reproduction of cellular arrangements and restoration of tissues. The principle is scratching a diamond lapping film that gives uniaxial nanotopography on substrates. Cells are seeded to follow the geometric cue via contact guidance, resulting in highly oriented cell alignment. Remarkable biocompatibility is also demonstrated by the high cell viability on various substrates. In vivo studies in a wound healing model in mice show that the scratched film supports directed cell guidance on the nanostructure, with significantly reduced wound areas and inhibition of excessive collagen deposition. Rapid recovery of the epidermis and dermis is also shown by histological analyses, suggesting the potential application of the scratching technique as an advanced wound dressing material for effective tissue regeneration.

Antioxidants prevent particulate matter-induced senescence of lung fibroblasts.

Particulate matter (PM) contributes to human diseases, particularly lung disease; however, the molecular mechanism of its action is yet to be determined. Herein, we found that prolonged PM exposure induced the cellular senescence of normal lung fibroblasts via a DNA damage-mediated response. This PM-induced senescence (PM-IS) was only observed in lung fibroblasts but not in A549 lung adenocarcinoma cells. Mechanistic analysis revealed that reactive oxygen species (ROS) activate the DNA damage response signaling axis, increasing p53 phosphorylation, ultimately leading to cellular senescence via an increase in p21 expression without affecting the p16-pRB pathway. A549 cells, instead, were resistant to PM-IS due to the PM-induced ROS production suppression. Water-soluble antioxidants, such as vitamin C and N-Acetyl Cysteine, were found to alleviate PM-IS by suppressing ROS production, implying that antioxidants are a promising therapeutic intervention for PM-mediated lung pathogenesis.

TPX2 prompts mitotic survival via the induction of BCL2L1 through YAP1 protein stabilization in human embryonic stem cells.

Genetic alterations have been reported for decades in most human embryonic stem cells (hESCs). Survival advantage, a typical trait acquired during long-term in vitro culture, results from the induction of BCL2L1 upon frequent copy number variation (CNV) at locus 20q11.21 and is one of the strongest candidates associated with genetic alterations that occur via escape from mitotic stress. However, the underlying mechanisms for BCL2L1 induction remain unknown. Furthermore, abnormal mitosis and the survival advantage that frequently occur in late passage are associated with the expression of BCL2L1, which is in locus 20q11.21. In this study, we demonstrated that the expression of TPX2, a gene located in 20q11.21, led to BCL2L1 induction and consequent survival traits under mitotic stress in isogenic pairs of hESCs and human induced pluripotent stem cells (iPSCs) with normal and 20q11.21 CNVs. High Aurora A kinase activity by TPX2 stabilized the YAP1 protein to induce YAP1-dependent BCL2L1 expression. A chemical inhibitor of Aurora A kinase and knockdown of YAP/TAZ significantly abrogated the high tolerance to mitotic stress through BCL2L1 suppression. These results suggest that the collective expression of TPX2 and BCL2L1 from CNV at loci 20q11.21 and a consequent increase in YAP1 signaling promote genome instability during long-term in vitro hESC culture.

Virus detection of measles-negative cases in Gyeonggi Province, South Korea, from 2017 to 2019.

To investigate viruses in measles-negative cases, 221 measles-suspected samples collected in Gyeonggi Province, South Korea were tested using a real-time PCR assay. Rubella virus was not detected. However, 11 cases of parvovirus B19 (5.0%), 47 cases of human herpesvirus 6 (21.3%), 25 cases of human herpesvirus 7 (11.3%), and one case of co-infection with parvovirus B19 and human herpesvirus 7 were confirmed, as were eight cases of co-infection with human herpesvirus 6 and human herpesvirus 7. This study showed that parvovirus B19, human herpesvirus 6, and human herpesvirus 7 should be considered by physicians for the diagnosis of measles-suspected patients.

Elucidation of divergent desaturation pathways in the formation of vinyl isonitrile and isocyanoacrylate.

Two different types of desaturations are employed by iron- and 2-oxoglutarate-dependent (Fe/2OG) enzymes to construct vinyl isonitrile and isocyanoacrylate moieties found in isonitrile-containing natural products. A substrate-bound protein structure reveals a plausible strategy to affect desaturation and hints at substrate promiscuity of these enzymes. Analogs are synthesized and used as mechanistic probes to validate structural observations. Instead of proceeding through hydroxylated intermediate as previously proposed, a plausible carbocation species is utilized to trigger C=C bond installation. These Fe/2OG enzymes can also accommodate analogs with opposite chirality and different functional groups including isonitrile-(D)-tyrosine, N-formyl tyrosine, and phloretic acid, while maintaining the reaction selectivity.

Utility of Diffusion and Magnetization Transfer MRI in Cervical Spondylotic Myelopathy: A Pilot Study.

Diffusion tensor imaging (DTI) and magnetization transfer (MT) magnetic resonance imaging (MRI) can help detect spinal cord pathology, and tract-specific analysis of their parameters, such as fractional anisotropy (FA), mean diffusivity, axial diffusivity (AD), radial diffusivity (RD) and MT ratio (MTR), can give microstructural information. We performed the tract-based acquisition of MR parameters of three major motor tracts: the lateral corticospinal (CS), rubrospinal (RuS) tract, and lateral reticulospinal (RS) tract as well as two major sensory tracts, i.e., the fasciculus cuneatus (FC) and spinal lemniscus, to detect pathologic change and find correlations with clinical items. MR parameters were extracted for each tract at three levels: the most compressed lesion level and above and below the lesion. We compared the MR parameters of eight cervical spondylotic myelopathy patients and 12 normal controls and analyzed the correlation between clinical evaluation items and MR parameters in patients. RuS and lateral RS showed worse DTI parameters at the lesion level in patients compared to the controls. Worse DTI parameters in those tracts were correlated with weaker power grasp at the lesion level. FC and lateral CS showed a correlation between higher RD and lower FA and MTR with a weaker lateral pinch below the lesion level.

Structural and biochemical analyses of Bcl-xL in complex with the BH3 domain of peroxisomal testis-specific 1.

Antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins suppress apoptosis by interacting with proapoptotic regulators. They commonly contain a hydrophobic groove where the Bcl-2 homology 3 (BH3) domain of Bcl-2 family members or BH3 domain-containing non-Bcl-2 family proteins can be accommodated. Peroxisomal testis-specific 1 (Pxt1) was previously identified as a male germ cell-specific protein whose overexpression causes germ cell apoptosis and infertility in male mice. Sequence and biochemical analyses also showed that human Pxt1, which is composed of 134 amino acids and is longer than mouse Pxt1 consisting of only 51 amino acids, has a BH3 domain that interacts with antiapoptotic Bcl-2 proteins, including Bcl-2 and Bcl-xL. In this study, we determined the crystal structure of Bcl-xL bound to the human Pxt1 BH3 domain. The five BH3 consensus residues are well conserved in the human Pxt1 BH3 domain and make a critical contribution to the complex formation in a canonical manner. Structural and biochemical analyses also demonstrated that Bcl-xL interacts with the BH3 domain of human Pxt1 but not with that of mouse Pxt1, and that residues 76-83 of human Pxt1, absent in mouse Pxt1, play a pivotal role in the intermolecular binding to Bcl-xL. While Bcl-xL consistently colocalized with human Pxt1 in mitochondria, it did not do so with mouse Pxt1, when expressed in HeLa cells. Collectively, these data verified that human and mouse Pxt1 differ in their binding ability to the antiapoptotic regulator Bcl-xL, which might affect their functionality in controlling apoptosis.

Using fungible biosensors to evolve improved alkaloid biosyntheses.

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors; using RamR as a starting point, we evolve highly specific (>100-fold preference) and sensitive (half-maximum effective concentration (EC50) < 30 µM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine and noscapine. High-resolution structures reveal multiple evolutionary avenues for the malleable effector-binding site and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, a precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors enable the rapid engineering of pathways for therapeutic alkaloids.

RBFOX2-regulated TEAD1 alternative splicing plays a pivotal role in Hippo-YAP signaling.

Alternative pre-mRNA splicing is key to proteome diversity; however, the biological roles of alternative splicing (AS) in signaling pathways remain elusive. Here, we focus on TEA domain transcription factor 1 (TEAD1), a YAP binding factor in the Hippo signaling pathway. Public database analyses showed that expression of YAP-TEAD target genes negatively correlated with the expression of a TEAD1 isoform lacking exon 6 (TEAD1ΔE6) but did not correlate with overall TEAD1 expression. We confirmed that the transcriptional activity and oncogenic properties of the full-length TEAD1 isoform were greater than those of TEAD1ΔE6, with the difference in transcription related to YAP interaction. Furthermore, we showed that RNA-binding Fox-1 homolog 2 (RBFOX2) promoted the inclusion of TEAD1 exon 6 via binding to the conserved GCAUG element in the downstream intron. These results suggest a regulatory mechanism of RBFOX2-mediated TEAD1 AS and provide insight into AS-specific modulation of signaling pathways.

Machine learning-aided engineering of hydrolases for PET depolymerization.

Plastic waste poses an ecological challenge1-3 and enzymatic degradation offers one, potentially green and scalable, route for polyesters waste recycling4. Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste5, and a circular carbon economy for PET is theoretically attainable through rapid enzymatic depolymerization followed by repolymerization or conversion/valorization into other products6-10. Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temperature ranges, slow reaction rates and inability to directly use untreated postconsumer plastics11. Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives12 between 30 and 50 °C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week. FAST-PETase can also depolymerize untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymatic plastic recycling at the industrial scale.

Recyclable Periodic Nanostructure Formed by Sublimable Liquid Crystals for Robust Cell Alignment.

We demonstrate a facile method to fabricate a recyclable cell-alignment scaffold using nanogrooves based on sublimable liquid crystal (LC) material. Randomly and uniaxially arranged smectic LC structures are obtained, followed by sublimation and recondensation processes, which directly produce periodic nanogrooves with dimensions of a couple of hundreds of nanometers. After treatment with osmium tetroxide (OsO4), the nanogroove can serve as a scaffold to efficiently induce directed cell growth without causing cytotoxicity, and it can be used repeatedly. Together, various cell types are applied to the nanogroove, proving the scaffold's broad applicability. Depending on the nanotopography of the LC structures, cells exhibit different morphologies and gene expression patterns, compared to cells on standard glass substrates, according to microscopic observation and qPCR. Furthermore, cell sheets can be formed, which consist of oriented cells that can be repeatedly formed and transferred to other substrates, while maintaining its organization. We believe that our cell-aligning scaffold may pave the way for the soft material field to bioengineering, which can involve fundamentals in cell behavior and function, as well as applications for regenerative medicine.